A scenario for a genetically controlled fission of artificial vesicles E. Boenzli 1, M. Hadorn 1, M. G. Jørgensen 2, P. Eggenberger Hotz 3, M. M. Hanczyc 1, T. Yomo 4 (1) Center for Fundamental Living Technology (FLinT), Department of Physics and Chemistry, SDU, Denmark (2) Department of Biochemistry and Molecular Biology (BMB), SDU, Denmark (3) The Mærsk Mc-Kinney Møller Institute, SDU, Denmark (4) Department of Bioinformatics Engineering, Osaka University, Japan References (1) Noireaux V., Maeda Y.T., and Libchaber A. Development of an artificial cell, from self-organization to computation and self-reproduction Proc Natl Acad Sci U S A. 108(9): p (2) White J.M. Membrane fusion Science. 258(5084): p (3) Nomura F., Inaba T., Ishikawa S., Nagata M., Takahashi S., Hotani H., and Takiguchi K. Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion Proc Natl Acad Sci U S A. 101(10): p (4) (5) Pautot S., Frisken B.J., and Weitz D.A. Engineering asymmetric vesicles Proc Natl Acad Sci U S A. 100(19): p (6) Hadorn M. and Eggenberger Hotz P. DNA-mediated self-assembly of artificial vesicles PLoS One. 5(3): p. e9886. (7) Hadorn M. and Eggenberger Hotz P. Towards Personalized Drug Delivery: Preparation of an Encapsulated Multicompartment System. in 3rd International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC) Valencia, Spain. Acknowledgements We thank Steen Rasmussen (FLinT, SDU), Tommy Andersen (FLinT, SDU), Ann Zahle Andersen (BMB, SDU), Martin Røssel Larsen (BMB, SDU), and Davide De Lucrezia (Explora Srl, Venice) for their helpful contribution and support. Introduction The creation of artificial cells that bridge the living and non-living world represents one of the main scientific goals of the 21 st century. A major task in creating self- replicating artificial cells is controlling the expression of a vast number of proteins as a result of the genetic program of the cell as well as the nongenetic molecular self- organization processes. Consequently, internally controlled vesicle division represents one of the major challenges in the creation of artificial cells 1. Proteins on the surface of enveloped viruses induce the fusion of their own membrane with the host cell membrane to release their genome into the cell 2. Using an artificial system to analyze membrane fusion processes, it was shown that a small part of the viral envelope protein itself has the potential to induce fusion of liposomes if added externally 3. Scenario In the present work, we developed a scenario how a genetically controlled fission of vesicles may be achieved by the synthesis of small parts of the viral envelope protein within artificial vesicles. The integration of the viral proteins to the inner leaflet of vesicles (Fig. 1) may induce fission. The same mechanism in nature used to induce fusion of compartments may here be used here to induce division of vesicles. Principle of vesicle fission induced by short peptides (red/green). Procedure In the first step, the viral proteins are produced externally by processes of the organic chemistry. In the second step, they are produced internally by a cell-free transcription/ translation system enclosed in the vesicles (Fig. 2). Due to the association of the viral proteins with the inner leaflet of the vesicles, the membrane may become destabilized. A subsequent increase in the osmotic pressure in the bulk solution results in deflated vesicles and in repeated contacts of the inner membrane surfaces. This contact, in combination with the destabilization of the inner leaflet, may lead to a fusion of the opposing membrane areas, and as a consequence, to fission of the vesicles (Fig. 2). Vesicles represent an ideal tool to investigate enclosed systems hosting ongoing biochemical processes. Cell-free transcription/translation systems provide the minimal machinery to synthesize proteins in vitro 4 (red/green). The vesicle preparation protocol, i.e. the water-in-oil emulsion transfer method 5, offers perfect control over the composition of both the bulk solution and the intravesicular solution. The versatility of this vesicle preparation method was already increased by collaborators of this project 6, 7. Figure 1 Figure 2